DESCRIPTION: (Investigator's Abstract). The broad, long-term objective of this research is to determine the mechanisms which control the aggregation state of proteins in the lens. Aging-related increased aggregation and insolubilization of structural lens proteins, the crystallins, results in a progressive loss of lens transparency and leads to the development of senile cataract, the major cause of blindness worldwide. An important recent finding is that the major protein component of the lens, a-crystallin, is a small heat-shock protein and can act as a molecular chaperone by preventing aggregation of other damaged proteins. The major goal of this project is to determine the molecular mechanisms of the chaperone-like action of a-crystallin. The specific aims are: (1) To advance the understanding of the tertiary and quaternary structure of a-crystallin; (2) To determine the conformational specificity of a-crystallin as a molecular chaperone, i.e. to identify specific conformational states of non-native proteins in which these proteins can be recognized by a-crystallin; (3) To determine which structural/conformational features of a-crystallin are essential for the chaperone activity and to identify the chaperone binding site; (4) To determine whether post-translational modifications of a-crystallin that impair the chaperone function can be correlated with specific changes in structural and/or conformational properties of the protein. The experimental design of this project constitutes a combination of biophysical, spectroscopic and biochemical approaches with molecular cloning and site-directed mutagenesis. The methods of genetic engineering will be employed for 13C-labeling of human a-crystallin as well as for site-specific incorporation of spectroscopic probes. These genetically engineered proteins will be used to study the structure of a-crystallin and the mode of a-crystallin interaction with other lens proteins by the emerging techniques of mutagenesis-assisted fluorescence spectroscopy and isotope-edited Fourier-transform infrared spectroscopy. Other methods to be used in this study include conventional Fourier- transform infrared spectroscopy, hydrophobic photolabelling/peptide mapping and circular dichroism spectroscopy.